陳桂芳，梁志強(qiáng)，李根喜,

1.南京大學(xué)生物化學(xué)系，醫(yī)藥生物技術(shù)國家重點(diǎn)實(shí)驗(yàn)室，南京 210093；

2.上海大學(xué)生命科學(xué)學(xué)院，上海 200444

納米材料用于構(gòu)建新型電化學(xué)生物傳感器的研究進(jìn)展

陳桂芳1，梁志強(qiáng)2，李根喜1,2

1.南京大學(xué)生物化學(xué)系，醫(yī)藥生物技術(shù)國家重點(diǎn)實(shí)驗(yàn)室，南京 210093；

2.上海大學(xué)生命科學(xué)學(xué)院，上海 200444

生物傳感器是目前生命科學(xué)及臨床醫(yī)學(xué)測(cè)試方法研究中最為活躍的領(lǐng)域之一，電化學(xué)生物傳感器是其中一個(gè)重要分支，已廣泛用于臨床、工業(yè)、環(huán)境和農(nóng)業(yè)分析等領(lǐng)域。進(jìn)入21世紀(jì)，納米科技的迅猛發(fā)展為新型生物傳感器的研制提供了難得的機(jī)遇。本文簡(jiǎn)要介紹和總結(jié)近年來基于蛋白質(zhì)和納米材料發(fā)展新型電化學(xué)生物傳感器的研究進(jìn)展，著重探討納米材料在構(gòu)建新型電化學(xué)生物傳感器中的應(yīng)用。

蛋白質(zhì)；納米材料；電化學(xué)生物傳感器；酶?jìng)鞲衅?；免疫傳感?/p>

0 引 言

生物傳感器一詞已經(jīng)被廣泛用來描述一類用以監(jiān)控生命體系或與之相關(guān)聯(lián)的生物基元的器件。其完整的概念可以認(rèn)為是由作為識(shí)別元件的固定化的生物敏感材料 (包括酶、抗體、微生物、細(xì)胞、組織、核酸等生物活性物質(zhì))，以及作為物理換能器的適當(dāng)?shù)男盘?hào)轉(zhuǎn)換器 (如氧電極、光敏管、場(chǎng)效應(yīng)管、壓電晶體等)所組成的體系。當(dāng)待測(cè)物質(zhì)與分子識(shí)別元件發(fā)生特異性的結(jié)合后，所產(chǎn)生的復(fù)合物信號(hào) (如光、熱、電等)通過信號(hào)轉(zhuǎn)換器轉(zhuǎn)變成光、電等可輸出信號(hào)，從而達(dá)到對(duì)待測(cè)物質(zhì)進(jìn)行分析檢測(cè)的目的。從識(shí)別元件來看，蛋白質(zhì) (主要包括酶、抗體)相比其他材料而言，具有功能的多樣性、對(duì)特定底物識(shí)別的專一性，以及分子本身的可操控性等不可比擬的優(yōu)點(diǎn)，因此成為目前最為常用的識(shí)別元件。而從信號(hào)轉(zhuǎn)換來看，電化學(xué)技術(shù)由于具備靈敏、快速、成本低廉、檢測(cè)裝置輕便、低能耗且易于微型化和集成化等優(yōu)點(diǎn)，已成為生物傳感器領(lǐng)域最為活躍的檢測(cè)手段[1,2]。

進(jìn)入21世紀(jì)，隨著納米技術(shù)的飛速發(fā)展，納米材料在醫(yī)學(xué)成像、疾病診斷、藥物傳輸、基因治療等多個(gè)領(lǐng)域顯示了巨大的優(yōu)勢(shì)。對(duì)于生物傳感領(lǐng)域而言，納米材料在光學(xué)性能、電學(xué)性能、磁學(xué)性能、力學(xué)性能和化學(xué)活性等方面表現(xiàn)出的獨(dú)特性能使其成為很好的換能器元件；另一方面，生物傳感器中的分子運(yùn)作本身就是基于納米層次，納米材料的參與可以將其優(yōu)良的性能很好地整合到分子運(yùn)作中，從而改進(jìn)甚至革新分子運(yùn)作的體系。鑒于以上特點(diǎn)，納米材料在生物傳感器中的應(yīng)用引起了越來越多科研工作者的興趣。本文從納米材料的角度綜述了近年來基于蛋白質(zhì)與納米材料發(fā)展新型電化學(xué)生物傳感器方面的研究進(jìn)展。

1 金屬納米材料

金屬納米材料良好的電子傳遞性能使其成為電化學(xué)生物傳感器中最為常用的納米材料之一，其中尤以納米金的應(yīng)用最為廣泛。納米金制備簡(jiǎn)單、性狀穩(wěn)定、生物相容性良好，而且易于進(jìn)行表面化學(xué)修飾，因此，利用納米金與生物分子進(jìn)行組裝并介導(dǎo)電子傳遞，是構(gòu)建電化學(xué)生物傳感器的良好方案。

Willner課題組[3]于2003年在Science上發(fā)表文章介紹了納米金的經(jīng)典應(yīng)用。他們將葡萄糖氧化酶 (glucose oxidase，GOD)的輔基FAD修飾到納米金表面，再通過雙巰基化合物的連接將FAD修飾的納米金固定到金電極上。當(dāng)引入脫輔基的GOD后，由于FAD與酶的特異結(jié)合，完整的GOD可以最大限度地保持其原有結(jié)構(gòu)和活性而組裝到電極表面，從而催化葡萄糖的氧化。通過監(jiān)測(cè)催化過程中FAD經(jīng)納米金介導(dǎo)傳遞到電極表面的電化學(xué)信號(hào)，可以對(duì)底物葡萄糖進(jìn)行定量分析。此后他們又利用納米金與聚陰離子組成的“微桿”介導(dǎo)得到了溶液中GOD的電化學(xué)響應(yīng)，從而實(shí)現(xiàn)葡萄糖的高靈敏檢測(cè) (圖1)[4]。結(jié)果顯示，納米金的使用一方面使得GOD與電極間的電子傳遞速率提高了25倍，另一方面，“微桿”結(jié)構(gòu)所提供的巨大的比表面積使得即使在溶液狀態(tài)下也能獲得GOD的電化學(xué)信號(hào) (圖2)。近年來，基于納米金與GOD所構(gòu)建的各種葡萄糖生物傳感器層出不窮，并發(fā)展出了一系列優(yōu)秀的實(shí)驗(yàn)設(shè)計(jì)方案。比如，Sun等發(fā)展了GOD在納米金表面的共價(jià)固定[5]以及基于層層組裝技術(shù) (layer-by-layer，LBL)的多層設(shè)計(jì)[6]，Ju等[7]發(fā)展了摻入GOD和納米金顆粒的碳糊電極，Chen等[8]發(fā)展了殼聚糖和納米金的聯(lián)合運(yùn)用，還有許多實(shí)驗(yàn)方案不在此一一列舉[9～16]。

圖1 葡萄糖氧化酶存在條件下，通過聚苯胺-納米金微桿(A)和聚苯胺-聚苯乙烯磺酸鹽微桿(B)介導(dǎo)的電化學(xué)催化氧化葡萄糖的示意圖[4]Fig.1 Bioelectrocatalytic oxidation of glucose in the presence of GOx mediated by Polyaniline/Au-nanoparticles composite micro-rods(A)and polyaniline/polystyrene sulfonate composite micro-rods(B)[4]

圖2 葡萄糖氧化酶存在下通過微桿介導(dǎo)的葡萄糖的電化學(xué)催化氧化[4](A)聚苯胺-納米金微桿介導(dǎo)的不同濃度葡萄糖氧化的循環(huán)伏安圖；(B)聚苯胺-聚苯乙烯磺酸鹽微桿介導(dǎo)的不同濃度葡萄糖氧化的循環(huán)伏安圖。a:0 mmol/L;b:10 mmol/L;c:60 mmol/LFig.2 Bioelectrocatalytic oxidation of glucose by solubilized GOx,1 mg mL-1,and mediated by the microstructured redox-active rod assemblies[4](A) Cyclicvoltammogramsrecorded in thepresenceof the microstructured assemblies composed of PAn/Au-NPs and different concentrations of glucose; (B)Cyclic voltammograms recorded in the presence of the microstructured assemblies composed of PAn/PSS and different concentrations of glucose.a:0 mmol/L;b:10 mmol/L;c:60 mmol/L

除了基于GOD的葡萄糖傳感器外，在基于辣根過氧化物酶 (horseradish peroxidase，HRP)的H2O2傳感器[17～28]、基于次黃嘌呤氧化酶 (xanthine oxidase，XOD)的次黃嘌呤/黃嘌呤傳感器[29～31]、基于乙醇脫氫酶 (alcohol dehydrogenase，ADH)的乙醇傳感器[32]、基于乳酸脫氫酶（lactate dehydrogenase，LDH）的乳酸傳感器[33]、基于乙酰膽堿酯酶的含磷殺蟲劑傳感器[34,35]、基于膽固醇氧化酶的膽固醇傳感器[36]，以及基于酪氨酸酶的酚類傳感器[37,38]等設(shè)計(jì)中，納米金的應(yīng)用同樣取得了很好的效果。此外，生理?xiàng)l件下并不作為酶的一些氧化還原蛋白，比如血紅蛋白 (hemoglobin，Hb)、肌紅蛋白 (myoglobin，Mb)以及細(xì)胞色素c(cytochrome c，cyt c)，經(jīng)過與納米金在電極表面共修飾組裝后也可表現(xiàn)出一定的酶活性，因而可用于構(gòu)建檢測(cè)H2O2、NO等底物的生物傳感器[39～45]。

納米金的另一個(gè)主要應(yīng)用是參與構(gòu)建基于抗原-抗體識(shí)別的電化學(xué)免疫傳感器。圖3顯示的是一個(gè)典型的納米金參與的免疫傳感器的示意圖。相比經(jīng)典的酶聯(lián)免疫傳感器——ELISA，納米金更為穩(wěn)定，在復(fù)雜的檢測(cè)環(huán)境不易降解和變性。更重要的是，通過與其他納米材料聯(lián)用，可以實(shí)現(xiàn)多靶標(biāo)的檢測(cè)?；诩{米金的這些優(yōu)良特性，近年來，涌現(xiàn)出眾多基于納米金的免疫傳感器[46～64]。比如，蔣健暉等[65]以人免疫球蛋白G(h IgG)為模型分析物，使固定化抗體與分析目標(biāo)物h IgG及納米金標(biāo)記的h IgG抗體結(jié)合于電極表面，通過金沉積使電極表面的納米金顆粒直徑增大，并通過金增強(qiáng)表面吸附伏安分析實(shí)現(xiàn)了h IgG的高靈敏定量免疫檢測(cè)。袁若[66]等通過LBL組裝將多層辣根過氧化物酶/納米金及L-半胱氨酸等材料組裝到電極表面，實(shí)現(xiàn)對(duì)甲胎蛋白抗體的固定，研制出用于檢測(cè)甲胎蛋白抗原(alpha-fetoprotein，AFP)的免疫傳感器。信號(hào)標(biāo)記對(duì)于免疫傳感器而言是煩瑣的一步，Yu等[67]使用納米金發(fā)展了無標(biāo)記的IgE檢測(cè)，并有望成為通用的免疫傳感設(shè)計(jì)。

圖3 (A)一抗通過靜電作用或共價(jià)連接固定到碳電極表面；(B)目標(biāo)蛋白（抗原）被固定的一抗識(shí)別；(C)金屬納米顆粒標(biāo)記的二抗結(jié)合抗原形成“三明治”結(jié)構(gòu)，從而可被電化學(xué)手段定量檢測(cè)到[46]Fig.3 (A)Primary antibodies are immobilized on the carbon-electrode surfaces through electrostatic attraction or covalent-binding reactions;(B)Target protein(antigen)is recognized by the primary antibody immobilized on the surface;(C)Metal nanoparticle-labeled secondary antibody binds to the antigen through its unique epitope,forming a sandwich-type complex on the surface.The accumulation of metal nanoparticles on the surface can be quantified using electrochemical-measurement techniques[46]

綜合來看，納米金用于構(gòu)建以蛋白質(zhì)為識(shí)別元件的電化學(xué)生物傳感器主要具備以下幾個(gè)優(yōu)勢(shì)：1)作為金屬材料，納米金是電子的良導(dǎo)體，可以最大限度地促進(jìn)電子傳遞，從而實(shí)現(xiàn)低電位下的電分析，以避免高電位中的噪音和干擾。2)作為納米材料，其大的比表面積十分有利于蛋白質(zhì)以及電化學(xué)信號(hào)探針等分子在電極表面的大量固定，從而有效地提高信號(hào)強(qiáng)度并實(shí)現(xiàn)信號(hào)放大。3)納米金具有良好的生物相容性，即使進(jìn)行在體實(shí)驗(yàn)也未發(fā)現(xiàn)對(duì)細(xì)胞有明顯的毒副作用；在分子層面，這種良好的生物相容性還可為蛋白質(zhì)提供良好的微環(huán)境，從而保持蛋白質(zhì)的生物活性。4)納米金可以很方便的通過靜電作用或共價(jià)作用進(jìn)行表面的化學(xué)修飾，從而為多樣化的蛋白質(zhì)表面固定方案打下基礎(chǔ)。

納米銀和納米鉑是另外兩種在電化學(xué)生物傳感器構(gòu)建中較為常用的金屬納米材料[68～75]。由于與納米金的性質(zhì)相似，基于這兩種納米材料的傳感器設(shè)計(jì)與基于納米金的傳感器設(shè)計(jì)有異曲同工之妙，在此不一一贅述。

2 碳納米管

自從1991年首次被報(bào)道以來，碳納米管 (carbon nanotubes，CNTs)可以說是被研究得最多的納米材料。對(duì)其理化性質(zhì)的深入了解，以及對(duì)其功能的不斷開拓，為其在生物傳感器構(gòu)建中的廣泛應(yīng)用打下了基礎(chǔ)。與納米金一樣， CNTs同樣也具備極好的電子傳遞能力、蛋白質(zhì)的高負(fù)載能力以及良好的生物相容性，而且，由于CNTs本身的物質(zhì)基礎(chǔ)就是碳，因此其功能化將更為方便和多樣。此外，由于CNTs為一維納米材料，意味著CNTs在電極表面的組裝將呈現(xiàn)網(wǎng)絡(luò)狀，因此，非常有利于蛋白質(zhì)的固定。

Davis等[76]精細(xì)地描繪了GOD在單壁碳納米管 (single-walled nanotubes，SWNTs)上的組裝 (圖4)。他們通過氧化SWNTs，使之表面帶上充裕的活性基團(tuán)，一方面有助于SWNTs在溶液中的分散，另一方面也給GOD的組裝提供了便利條件。結(jié)果顯示，即使不加入EDC、NHS等共價(jià)連接的活化劑，GOD也能在SWNTs表面很強(qiáng)地吸附并保持活性。他們將此吸附有GOD的SWNTS組裝到玻碳電極上，得到了良好的GOD響應(yīng)，其信號(hào)強(qiáng)度是沒有SWNTs參與時(shí)的10倍，顯示出SWCNTs在酶負(fù)載和能量轉(zhuǎn)換上都具有良好的效果。

圖4 (A)葡萄糖氧化酶修飾的單壁碳納米管的原子力顯微鏡照片；(B)葡萄糖氧化酶-單壁碳納米管共修飾的玻碳電極在無（曲線c）或有（曲線b）二茂鐵一元酸，以及加入50 mmol/L葡萄糖（曲線a）后的循環(huán)伏安圖[76]Fig.4 (A)Amplitude AFM image of a glucose oxidase-modified SWNT.Scale bar=200 nm.(B)Voltammetric response of a GOX-SWNT-modified GC electrode in the absence(curve c)and presence(curve b)of 0.5 mmol/L ferrocenemonocarboxylicacid (FMCA). Thecatalyticresponse(curvea) isobserved on theaddition of 50 mmol/L glucose[76]

除了在電極表面的平鋪組裝外，CNTs還可以通過化學(xué)氣相沉積 (chemical vapor deposition，CVD)直接在電極表面垂直生長。Fang等[77]運(yùn)用這種方法在電極表面生長CNTs，并利用 (4-羧苯基)重氮鹽在CNTs表面的電化學(xué)還原對(duì)CNTs進(jìn)行了功能化，從而共價(jià)連接Hb，結(jié)果發(fā)現(xiàn)Hb具有良好的電化學(xué)響應(yīng)，并可用于構(gòu)建H2O2傳感器（圖5）。Wisitsoraat等[78]也利用這種方法在金覆蓋的SiO2/Si基底上生長了CNTs，然后通過電化學(xué)聚合在CNTs表面固定聚苯胺 (polyaniline，PANI)和膽固醇氧化酶，利用循環(huán)伏安法對(duì)底物膽固醇進(jìn)行了檢測(cè)，取得了良好的結(jié)果。

目前，基于以上兩種方式，利用CNTs構(gòu)建酶?jìng)鞲衅鱗79～97]和免疫傳感器[98～106]的報(bào)道數(shù)量眾多，在此不一一列舉。需要提出的是，由于CNTs難以具備納米金那樣良好的形態(tài)分布，因此對(duì)有序的表面組裝提出了挑戰(zhàn)。另外，大多數(shù)蛋白質(zhì)的尺寸都屬于零維的納米級(jí)，因此在一維的CNTs表面組裝相對(duì)而言缺少靈活性。出于這些考慮，將CNTs與零維的納米顆粒，如納米金、納米鉑等聯(lián)合運(yùn)用，在一定程度上可以克服兩者在某些方面的缺陷，因而也是傳感器構(gòu)建中的良好策略[107～113]。

圖5 (A)碳納米管陣列的掃描電子顯微鏡照片；(B)血紅蛋白-碳納米管陣列修飾電極在加入不同濃度過氧化氫前后的循環(huán)伏安圖[77]Fig.5 (A)SEM images of ACNTs;(B)Cyclic voltammograms of the Hb-ACNTs electrode in PBS(pH 7.0)before(a)and after the addition of 160,360,560,760,960 mmol/L H2O2(curve b to f).Scan rate:40 mV/s[77]

3 納米氧化物

除了具備納米材料共有的一些性質(zhì)外，納米氧化物還依材料的不同具備一些特殊的效應(yīng)，比如納米Fe3O4的磁效應(yīng)、納米TiO2的光電效應(yīng)等。而這些效應(yīng)在新型生物傳感器的構(gòu)建中可以產(chǎn)生一些意想不到的效果。在這一部分，我們將著重就納米氧化物特殊效應(yīng)的應(yīng)用進(jìn)行總結(jié)和回顧。

磁性納米顆粒現(xiàn)在已被廣泛用于分離富集、靶向運(yùn)輸?shù)确矫娴难芯浚谏飩鞲蓄I(lǐng)域，磁效應(yīng)的應(yīng)用還不廣泛。Willner等[114]利用納米Fe3O4首次實(shí)現(xiàn)了電化學(xué)催化的磁控制，如圖6所示，他們首先合成了能夠穩(wěn)定分散于有機(jī)溶劑中的磁性納米Fe3O4，并構(gòu)建了一個(gè)雙層的電解液，上層為有機(jī)溶劑甲苯，下層為水溶液。當(dāng)磁場(chǎng)處于上方時(shí)，F(xiàn)e3O4分散在甲苯中，下方的GOD可以催化葡萄糖的氧化并得到電信號(hào)；但當(dāng)磁場(chǎng)轉(zhuǎn)移到電極下方，F(xiàn)e3O4將被吸引下來覆蓋住電極表面，從而阻礙GOD與電極的電子傳遞，失去了電驅(qū)動(dòng)的GOD便無法進(jìn)行催化，由此，通過轉(zhuǎn)換磁場(chǎng)方向即可非常方便地控制酶催化反應(yīng)進(jìn)程。

納米TiO2是另一種具有特殊效應(yīng) (光電效應(yīng))的納米材料，由于具有極強(qiáng)的紫外線屏蔽能力和很高的表面活性，納米TiO2已經(jīng)被大量用于污水處理、消毒殺菌，以及在化妝品和涂料中防紫外線侵蝕。作者所在實(shí)驗(yàn)室曾利用納米TiO2這種特有的性質(zhì)構(gòu)建了高靈敏的H2O2傳感器 (圖7)[115]。一般來講，蛋白質(zhì)在紫外線照射下很容易變性，但是我們利用納米TiO2與Hb共修飾后發(fā)現(xiàn)，一定時(shí)間的紫外線照射后，Hb不僅沒有失活，其催化活性反而大大提高，電化學(xué)檢測(cè)H2O2的靈敏度提高了3倍，檢測(cè)限降低了兩個(gè)數(shù)量級(jí)。這一方面得益于TiO2對(duì)紫外線的屏蔽保護(hù)了Hb，另一方面，TiO2在紫外線激發(fā)下產(chǎn)生的激發(fā)態(tài)活性物質(zhì)促進(jìn)了Hb催化性能的提高。作者所在實(shí)驗(yàn)室在后續(xù)的研究中發(fā)現(xiàn)納米ZnO也具有類似的效用[116]。

圖6 (A)磁性納米顆粒遠(yuǎn)離電極從而激活二茂鐵介導(dǎo)的葡萄糖的電化學(xué)催化氧化；(B)電極表面被疏水的磁性納米顆粒覆蓋從而阻礙了電化學(xué)催化的物質(zhì)擴(kuò)散過程[114]Fig.6(A)The magnetic nanoparticles are retracted from the electrode surface,which is activated toward the ferrocene-mediated bioelectrocatalytic oxidation of glucose;(B)The electrode surface is blocked by the hydrophobic magnetic nanoparticles toward the diffusional bioelectrocatalytic process,while the surface-confined ferrocene is electrochemically active[114]

圖7 二氧化鈦納米顆粒與血紅蛋白共修飾電極的示意圖[115]Fig.7 Scheme of TiO2nanoparticle/Hb co-modified electrode[115]

4 量子點(diǎn)

量子點(diǎn)作為熒光標(biāo)記物，已經(jīng)被廣泛用于熒光示蹤。在電化學(xué)生物傳感器領(lǐng)域，量子點(diǎn)同樣有著特殊的用途。以金屬硫/硒/碲化物 (Zn/Cd/Pb-S/Se/Te)等為代表的量子點(diǎn)，一方面是很好的生物標(biāo)記材料，另一方面，其中的金屬離子Zn2+、Cd2+、Pb2+可用于陽極溶出伏安法檢測(cè)，從而提供電化學(xué)信號(hào)。

Wang等[117]利用三種量子點(diǎn) (ZnS、CdS以及PbS)分別標(biāo)記三種抗體，并利用磁性納米顆粒進(jìn)行分離富集，通過溶出伏安法檢測(cè)Zn2+、Cd2+、Pb2+在不同電位下的峰電流，可以同時(shí)檢測(cè)三種抗原的存在 (圖8)。其他一些課題組也利用量子點(diǎn)的這種性質(zhì)進(jìn)行了一些傳感設(shè)計(jì)[118～120]。

圖8 基于不同無機(jī)納米顆粒的多蛋白的電檢測(cè)原理[117](A)引入抗體修飾的磁珠；(B)抗原結(jié)合到磁珠表面的抗體上；(C)納米顆粒標(biāo)記的二抗被磁珠捕獲；(D)對(duì)納米顆粒進(jìn)行電化學(xué)溶出分析Fig.8 Multi-protein electrical-detection protocol based on different inorganic nanoparticle(NP)tracers[117](A)Introduction of antibody-modified magnetic beads;(B)Binding of antigens to antibodies on the magnetic beads;(C)Capture of the NP-labeled secondary antibodies;(D)Dissolution of NPs and electrochemical stripping detection

總的來說，基于量子點(diǎn)的電化學(xué)傳感設(shè)計(jì)具有以下一些優(yōu)點(diǎn)：1)由于近年來量子點(diǎn)相關(guān)技術(shù)的日趨成熟，用于電化學(xué)檢測(cè)的量子點(diǎn)的制備以及功能化比較方便，有商品化的產(chǎn)品可供選擇；2)不同的量子點(diǎn)可以提供不同的電化學(xué)信號(hào)，這為多組分的分析提供了條件；3)溶出伏安法是基于金屬離子的氧化還原分析，其理論檢測(cè)限可達(dá)到飛摩爾水平，這為痕量檢測(cè)打下了基礎(chǔ)。

5 復(fù)合納米材料

不同的納米材料各自具備一定的特性，在電化學(xué)生物傳感器的設(shè)計(jì)中使用單一的材料難以充分發(fā)揮納米材料的性能，因此，同時(shí)使用多種納米材料成為一個(gè)解決方案。一種思路是首先合成兩種或多種納米材料，然后在傳感器的構(gòu)建中同時(shí)或在不同階段分別運(yùn)用，比如圖6所介紹的量子點(diǎn)與磁性納米顆粒的運(yùn)用；另一種思路則是在納米材料的合成階段將不同的材料進(jìn)行組裝，即合成復(fù)合納米材料，將不同納米材料的特性整合到一個(gè)納米復(fù)合體中。一個(gè)很好的例子是CNTs與金屬納米顆粒復(fù)合的材料，另一個(gè)例子則是合成核/殼結(jié)構(gòu)的納米顆粒，而且這種做法目前更為常見。

Yu等[121]合成了MgFe2O4@SiO2核/殼納米顆粒，由SiO2外殼提供良好的酪氨酸酶組裝界面，由磁性的MgFe2O4內(nèi)核實(shí)現(xiàn)蛋白在電極表面的磁控富集。由此構(gòu)建的電化學(xué)生物傳感體系可以對(duì)酶底物苯酚實(shí)現(xiàn)良好的檢測(cè)。這種核/殼納米顆粒一方面保持了磁性納米顆粒的磁效應(yīng)，另一方面又為蛋白質(zhì)提供了良好的組裝界面和微環(huán)境，因而成功地綜合了兩種納米材料的優(yōu)良特性。Zhu等[122]則充分利用AgCl和PANI的特性合成了AgCl@PANI納米顆粒，一方面AgCl外殼克服了PANI在水相中難以分散的缺點(diǎn)，并為后續(xù)的電化學(xué)信號(hào)獲取提供了良好的電子傳遞界面，另一方面，PANI為傳感器在堿性條件下的使用提供了條件，并有效地防止了抗壞血酸的氧化，他們利用此復(fù)合納米材料構(gòu)建了用于檢測(cè)多巴胺的電化學(xué)生物傳感器。此外， Calvo、Zeng等課題組[123～127]也開展了一系列基于核/殼納米顆粒的傳感器研究工作。

6 展 望

納米科技與電化學(xué)技術(shù)的結(jié)合和相互滲透，為基于蛋白質(zhì)構(gòu)建生物傳感器提供了重大的創(chuàng)新機(jī)遇和誘人的市場(chǎng)前景，各種具有優(yōu)良性能的納米材料在生物傳感器構(gòu)建中的應(yīng)用，不僅大大提升了生物傳感器的性能，還拓寬了生物傳感器的適用范圍，為其在臨床檢測(cè)、食品安全、環(huán)境監(jiān)測(cè)、醫(yī)療衛(wèi)生等領(lǐng)域的應(yīng)用開辟了新的道路。當(dāng)然，新技術(shù)出現(xiàn)的同時(shí)也面臨著新的挑戰(zhàn)，比如如何實(shí)現(xiàn)原位分析、如何構(gòu)建穩(wěn)定專一的電化學(xué)生物傳感芯片、如何建立健全的評(píng)價(jià)納米材料效用及安全性的標(biāo)準(zhǔn)體系，等等。隨著蛋白質(zhì)科學(xué)，以及納米技術(shù)、電化學(xué)技術(shù)、分子識(shí)別技術(shù)、表面固定技術(shù)等相關(guān)技術(shù)的不斷發(fā)展，我們相信這些問題會(huì)逐步得到解決，電化學(xué)生物傳感器也會(huì)獲得更大的發(fā)展，展示更為廣闊的應(yīng)用前景。

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This work was supported by grants from The National Science Fund for Distinguished Young Scholars(20925520)and Shanghai Science and Technology Committee(09DZ2271800)

Progress of Electrochemical Biosensors Fabricated with Nanomaterials

CHEN Guifang1,LIANG Zhiqiang2,LI Genxi1,2
1.Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology,Nanjing University,Nanjing 210093,China;
2.Laboratory of Biosensing Technology,School of Life Science,Shanghai University,Shanghai 200444,China

Feb 2,2010 Accepted：Apr 28,2010

LI Genxi,Tel：+86(25)83593596,E-mail：genxili@nju.edu.cn

Biosensorshave

more and more research interestsdue to the rapidlyincreased requirement for measurements in life sciences.Protein-based electrochemical biosensors,which are particular focused because of practical advantages,have been widely used in clinic,industry,environment,and agriculture.Due to the rapid development of nano Sci-Tech,many kinds of novel biosensors are fabricated.In this paper,we summarize the progress of protein-based electrochemical biosensors,with the emphasis on the discussion ofthe application ofnanomaterials to the developmentofelectrochemicalbiosensors fabricated with protein and nanomaterials.

Protein;Nanomaterial;Electrochemical biosensor;Enzyme biosensor;Immunosensor

2010-02-02；接受日期：2010-04-28

國家杰出青年科學(xué)基金項(xiàng)目(20925520)，上海市科學(xué)技術(shù)委員會(huì)項(xiàng)目(09DZ2271800)

李根喜，電話：(025)83593596，E-mail：genxili@nju.edu.cn

O657.1